1. Field of the Invention
The invention relates to the development of vaccines and specifically to the development of an improved method for antigen presentation. The invention further relates to the delivery of nucleic acids to cells via a particle.
2. Background Description
The aim of all vaccinations is to induce specific immunity that prevents microbial invasion, eliminates microbes that have already invaded the host, or neutralizes microbial toxins. Unfortunately, the development of effective protein subunit or peptide vaccines against intracellular pathogens has been hindered by major technological and conceptual inadequacies.
One such inadequacy is the inability to elicit a strong TH1 immune response. The response of the immune system to an antigen during vaccination can be somewhat variable depending on the size and composition of the disease specific antigen, and the particular adjuvant used to enhance the immune response. Potential responses include TH 1, TH2 or mixed (TH1/FH2) responses.
Cytokine profiles determine T cell regulatory and effector functions in immune responses. Cytokines play a role in directing the T cell response. Helper (CD4+) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including other T cells. Most mature CD4+T helper cells express one of two cytokine profiles: TH1 or TH2. TH1 cells secrete IL-2, IL-3, IFN-γ, TNF-β, GM-CSF and high levels of TNF-α. TH2 cells express IL-3, IL4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-α. The TH1 subset promotes delayed-type hypersensitivity, cell-mediated immunity, and immunoglobulin class switching to IgG2. The TH2 subset induces humoral immunity by activating B cells, promoting antibody production, and inducing class switching to IgG, and IgE.
Several factors have been shown to influence commitment to TH1 or TH2 profiles. The best characterized regulators are cytokines. IL-12 and IFN-γ are positive TH1 and negative TH2 regulators. IL-12 promotes IFN-γ production, and IFN-γ provides positive feedback for IL-12. IL4 and IL-10 appear to be required for the establishment of the TH2 cytokine profile and to down-regulate TH1 cytokine production; the effects of IL4 are in some cases dominant over those of IL-12. IL-13 was shown to inhibit expression of inflammatory cytokines, including IL-12 and TNF-α by LPS-induced monocytes, in a way similar to IL-4. The IL-12 p40 homodimer binds to the IL-12 receptor and antagonizes IL-12 biological activity; thus it blocks the pro-TH1 effects of IL-12.
A TH1 response is marked by mobilization of macrophages, B-cells antibody producing cells, professional antigen presenting cells (APCs), and cytotoxic T lymphocytes (CTL) for the phagocytic elimination of microbes. Further, a TH1 response induces the expression of IFN-γ. IFN-γ promotes the switching of B cells to isotypes such as IgG2 which fixes complement and promotes phagocytosis by macrophages. IFN-γ also activates the antimicrobial functions of macrophages. A TH1 immune response thus induces phagocyte-dependent host reactions that are important for the elimination of intracellular microbes, and it is therefore highly desirable for a vaccine to elicit a strong TH1 response.
Several studies have reported that the cytokine IL-12 plays a critical role in inducing antiviral effects in vivo by promoting the TH1-type immune response. IL-12 is mainly produced by activated antigen presenting cells (APCs) including macrophages, dendritic cells, and B cells, and is reported to augment antibody, CD4+, and CTL responses. IL-12 induces the maturation of type-1 TH cells into IFN-γ producing cells, promotes natural killer (NK) activity, and enhances CTL maturation. It is a heterodynamic cytokine consisting of two subunits, p35 and p40. The p35 subunit is constituitively expressed, while the p40 subunit is expressed only upon APC activation. It is therefore highly desirable for a vaccine to elicit the production of IL-12.
Protein subunit or peptide vaccines are often not immunologically active by themselves and must be administered with an adjuvant. Aluminum hydroxide (alum) is currently the only adjuvant approved for human use. An important disadvantage of alum is that it induces a TH2- rather than a TH 1-type immune response, and this may interfere with induction of CTL. Indeed, in mice immunized with recombinant Hepatitis B surface antigen (HBsAg), the addition of alum selectively blocked activation of CD8+CTL (Schirinbeck et al., 1994). Although not essential for protective immunity against HBV, CTL may nevertheless play an important role.
The use of alum has been linked to TH2-type diseases. The much higher prevalence of asthma (a TH2-type disease) in more highly developed nations may be linked to the high hygiene level and rapid treatment of childhood infections (Cookson and Moffatt, 1997). Early exposure to bacterial DNA pushes the immune system away from TH2- and towards a TH1-type response and this may account for the lower incidence of asthma in less developed countries, where there is a much higher frequency of upper respiratory infections during childhood. It would be an advantage to have available pediatric vaccines capable of re-establishing a TH1-type response, thereby reducing the incidence of asthma.
In order to elicit a strong TH1 response by an antigen which is deposited in the extracellular fluid (which is the case with most vaccines) very high concentrations of the antigen are necessary. This cannot be accomplished in a safe and efficacious manner. However, one solution to the problem is to deliver disease-specific antigens on the surface of a particle. Both macrophages and dendritic cells internalize particles into large vacuoles where exogenous antigens can be transferred to both the class I and class II presentation pathways. This exogenous class I presentation pathway is of considerable interest for vaccine development because it provides a means of eliciting CTL immunity with antigens that are deposited into the extracellular fluid. It has been demonstrated that when exogenous antigens are particulate in nature, they are presented 1,000 to 10,000-fold more efficiently than soluble antigens in both the class I and class II pathways (Harris et al., 1992; Griffiths et al., 1993; Schodel et al., 1994; Schirmbeck et al, 1995; and Raychaudhuri and Rock, 1998). One example of the use of particles to carry antigens is that of the human hepatitis B viral core antigen (HBcAg). It has been shown that human HBcAg is capable of serving as an effective carrier for foreign epitopes which have been chemically coupled to, or genetically engineered into, the protein sequence at several selected sites (Milich,1990; Schodel et al., 1992; Schodel et al., 1993; Schodel et al., 1994; Milich et al., 1995). Also, see, for example, the following Thornton et al.: U.S. Pat. Nos. 4,882,145; 4,882,145, and 5,143,726, which are incorporated herein by reference. The patents are directed toward the use of fusion proteins comprised of T cell stimulating regions of the human Hepatitis B viral (HBV) nucleocapsid protein linked to a polypeptide immunogen. However, one serious drawback to the use of human HBcAg as a vaccine carrier is that antibodies against HBcAg itself (anti-HBc) also develop in recipients of the vaccine. This is a problem because the detection of HBV infection in humans, and in blood which has been donated for use in transfusions, is by screening for anti-HBc in the blood. Therefore, widespread use of a vaccine based on human HBcAg would compromise the currently used hepatitis B screening system. The use of hepatitis B-based particles from some other species (e.g. woodchuck) would also pose the same problem since they are crossreactive with human HBcAg. It is estimated that approximately 500 million people are infected with HBV worldwide. In these individuals, the administration of a vaccine based on human HBcAg would be fruitless, since their immune system would likely attack and destroy the vaccine particles before they could exert their immunizing effect. It would therefore be advantageous to have available a particulate vaccine carrier which did not interfere with current HBV screening protocols.
Another major inadequacy of current vaccines is the inability to produce vehicles to deliver haptens that are effective in stimulating a specific immune response to “quasispecies” of the invading microbe. Quasispecies are progeny of the original microbe that develop during infection. The genetic structure of quasispecies has been altered by mutation, allowing these structurally similar microbes to escape elimination by the immune system and maintain the pathological condition. It would thus also be highly desirable to develop a hapten vehicle for use in vaccines that was capable of eliciting a protective response against a wide variety of structurally similar haptens such as those exhibited by genetically hypervariable microbes (e.g. viruses). Such a hapten vehicle could stimulate an immune response against the targeted microbe and quasispecies produced during infection.